The present application relates to a real-time PCR system. More specifically, the present application is concerned with a real-time PCR system for assaying gene expressions and the like.
In recent years, hybridization detection technologies led by DNA chips and DNA microarrays have been finding increasing practical applications. A DNA chip includes a wide variety of DNA probes integratedly immobilized on a surface of a substrate. By detecting, with this DNA chip, hybridizations on the surface of the substrate in the DNA chip, gene expressions in cells, a tissue or the like can be comprehensively assayed.
It has now become a standard method for the quantitative assay of trace nucleic acids to verify, by PCR (polymerase chain reaction), data acquired by such a microarray.
Real-time PCR can amplify DNA or the like to several hundred thousand-fold by continuously performing amplification cycles of “thermal denaturation→annealing with a primer→polymerase extension reaction”. Real-time PCR monitors the resulting PCR amplification products in real time to perform a quantitative assay of the trace nucleic acids.
According to real-time PCR, the PCR amplification products can be monitored using a dedicated system or the like with a thermal cycler and a fluorescence spectrophotometer combined together as an integral unit. As such systems, there are real-time PCR systems.
A real-time PCR system is a reaction and processing system that upon allowing amplification to proceed through reactions in a sample, irradiates exciting light and detects fluorescent signals in real time. It can be used as a detection system or the like to conduct genome DNA observations in medical care practice and gene analysis studies, including chemical reactions.
When labeling is conducted with a fluorescent dye to specify a double-stranded DNA of interest to be synthesized, for example, in a PCR method that amplifies DNAs, heating of the double-stranded DNA makes it possible to observe variations in intensity of fluorescence to be emitted from the florescent dye.
When only a target DNA of interest can be amplified with a high-specificity primer, for example, intercalation making use of “SYBRR™ GREEN I” (product of Molecular Probe, Inc.) can be used.
An intercalator, which is used emit fluorescence upon binding to a double-stranded DNA, binds to a double-stranded DNA synthesized by a PCR reaction, and emits fluorescence when exciting light is irradiated. By detecting the intensity of this fluorescence, the amount of the amplification product can be monitored. Intercalation, therefore, does not require design and synthesize a fluorescently-labeled probe specific to the target DNA, and can be easily used in the assay of various target DNAs.
Further, the probe method is used when there is a need to separately detect sequences which are very close to each other or when a multiplex detection is needed as in SNP typing. As one example of the probe method, there is the “TAQMAN™ PROBE” method (product of Applied Biosystems, Inc.) which uses as a probe an oligonucleotide modified at the 5′ terminal with a fluorescent material and at the 3′ terminal with a quencher material.
“TaqMan™ probe” specifically hybridizes with a template DNA in an annealing step. Due to the existence of a quencher on the probe, however, the emission of fluorescence is inhibited even when exciting light is irradiated. In an extension reaction step, the “TAQMAN PROBE” hybridized on the template is decomposed by the 5′→3′ exonuclease activity of TaqDNA polymerase, the fluorescent dye is liberated from the probe, the inhibition by the quencher is cancelled, and therefore, fluorescence is emitted. By measuring the intensity of this fluorescence, the amount of the resulting amplification product can be monitored.
A description will hereinafter be made of the principle of a quantitative assay of a gene expression level or abundance by real-time PCR in the above-described manner. Firstly, PCR is performed using as templates serially-diluted standard samples the concentrations of which are known. Numbers of cycles (threshold cycles: Ct values) required to each a certain constant amount of amplification product are then determined. A calibration line is then prepared by plotting these Ct values and initial DNA amounts along the abscissa and the ordinate, respectively.
With respect to a sample the concentration of which is unknown, a PCR reaction is also conducted under the same conditions to determine a Ct value. From this Ct value and the above-mentioned calibration line, the amount of the target DNA in the sample can be determined.
As techniques relating to the above-described real-time PCR, technologies on temperature control or the like are disclosed in Japanese Patent Laid-open Nos. 2003-298068 and 2004-025426.